3.3 離子交換法Ion exchange 3.3.3 緩衝液與層析系統Buffer system

3.3 離子交換法Ion exchange 3.3.3 緩衝液與層析系統Buffer system

3.3 離子交換法 Ion exchange
● 3.3.1 原理概述 Basic principles
離子與固相擔体帶電基團間的爭奪戰 (Ion wars)
● 3.2.2 交換介質 Exchange materials
是帶有電荷基團的多醣長鏈聚合物 (膠球)
● 3.3.3 緩衝液與層析系統 Buffer system
緩衝液的影響極大
● 3.3.4 管柱操作方法 Column operation
如何操作一支離子交換管柱
● 3.3.5 色層焦集法 Chromatofocusing
依蛋白質等電點之差異來進行分離
Juang RH (2005) EPA
■ 陰離子交換法 Anion Exchange
Sample
+
+ + +
+
Counter
ion
+
+ Solid
+
+ support
+
Anion
+
+
+
+
+ + Cation groups
Stationary phase
Ion exchange is an adsorption chromatography
Mobile
phase
+
Juang RH (2005) EPA
■ 質子可以吸著或脫離一基團
Proton： the smallest and most abundant particle in the living cell
controlling the pH and the charge property of a molecule
lone pair
electrons
Amino
H+
N H
H+
N H
H
Carboxylic
C
H
O H
O
O
C
O
H+
Ampholyte: a molecule contains both positively and negatively charged groups
Proton can enter or leave a functional group relatively freely
Juang RH (2005) EPA
■
選
擇
離
子
交
換
介
質
Buffer pH
Use buffer having
only one pH unit
difference to the
pI of target protein
10
9
8
7
pI
Cation exchange
+
6
5
4
3
0
Anion exchange
Net Charge of a Protein
Juang RH (2005) EPA
■ 陰離子交換膠體的 pH 變化
OHDonnan Effect
pH = 7
-
pI = 6
Counter
ion
+
pH = 5
The pH change of the microenvironment surrounding the ion exchange gel particle
Juang RH (2005) EPA
OH- pH = 8
+
OH+
Support +
+ OH+
■ 離子取代優先順序 Displacing order of ions
電荷高者
(higher charge)
離子大者
(larger ion)
濃度大者
(higher concentration)
change pH, NaCl gradient
++
+
+
+
+ + +
+ + + +
+
+ + + +
+
Key properties determining the order of ion replacement
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■
陰
離
子
交
換
法
的
分
離
範
圍
pH
kD
4
5
6
7
100
50
25
The approximate range of proteins isolated by chromatography
Juang RH (2005) EPA
■ 離子交換介質 Common ion exchange materials
Cation
Exchanger
Anion
Exchanger
Classification
Resin / Polystyrene
Dowex-1
Strong
Dowex-2
Weak
Dowex-3
IR-45
Strong Dowex-50
Weak
IRC-150
+
Glycan / Cellulose = X
TEAE-X
(QAE-X)
-NR3
+
-NHR2
-SO3
-COO
Q
-NR3
DEAE-X
+
-OCH2CH2NHR2
Phospho-X
-
+
Mono
bead
2-PO 4
S
-
CM-X -OCH COO
2
X = Sephadex, Sepharose, Sephacel or cellulose
Juang RH (2005) EPA
Pharmacia (1980) Separation News: 5
Cellulose
Sephacel
Monobead
● Homogeneous bead shape is critical to
its resolution and flow rate
Pharmacia (1991) Ion Exchange Chromatography – Principles and Methods p.24
■ 膠體的組成與外型 Support material and bead
■ 不同膠體的吸著容量有很大差異
Adapted from Pharmacia (1991) Ion Exchange Chromatography – Principles and Methods p.64
Lactalbumin
Albumin
Ferritin
DEAE-Sephadex A-25
191
31
2
DEAE-Sephadex A-50
10
102
1
DEAE-Sepharose CL-6B
45
115
4.3
DEAE-Sephacel
38
86
8.6
Buffer: 0.01 M Tris-HCl, pH 8.0
mg / mL gel
Each gel has different adsorption capacity toward different target proteins
■ 離子交換法預備試驗 Determine the conditions
Enzyme + Buffer + Gel
Incubation
Spin
Sup + Spindown
Sup
pH
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
10
20
30
40
50
60
70
80
90
100
Spindown
NaCl
pH = 7
0.15 M
NaCl
mg/mL
protein
Adapted from Pharmacia (1991) Ion Exchange Chromatography – Principles and Methods p.70
Capacity
■ 膠柱裝填方法 Packing column step by step
清洗膠體
Put on adaptor
Put on reservoir
Wash gel well
上清
預估體積
Supernatant
Estimate
gel volume
裝填膠體
檢查管柱
是否暢通
Pouring gel
smoothly
Check flow
rate of empty
column
Gel
沈積中
In progressing
緊
密
堆
積
已堆積
Sediment
靜置膠體
Gel stands o/n
緩衝液
平衡
Equilibrated
in buffer
溫度平衡
Temperature
equilibrated
1
2
Gel
暫停流洗
繼續流洗
Stop elution
Keep eluting
X
Gel should be equilibrated completely before packing
加壓流洗
Elute under
High pressure
Juang RH (2005) EPA
■
液
相
層
析
管
柱
系
統
梯度製造器
(2)
Gradient mixer
轉接器
Connector
幫浦
Pump
(1)
記錄器
adapter
緩衝液
Recorder
(3)
Buffer
(4)
管柱
A
Column
reservoir
膠體
Gel
監視器
Monitor
(5) 分劃收集器
Fraction collector
膠
體
裝
填
The whole family of liquid chromatography apparatus
Juang RH (2005) EPA
■ 鹽梯度的兩種方式 Two ways for making gradient
低限溶液
高限溶液
膠柱面
Gel surface
連續梯度
連續梯度
階段梯度
連續梯度
階段梯度
Continuous
Step-wise
Dead volume
Eliminate
dead
volume
Upper-limit
Lower-limit
兩種方式均可，各有特點。
Both methods have their specific applications
Pharmacia
Juang RH (2005) EPA
0.4
0.3
0.2
0.1
0
50
階段梯度
100
150
200
Step-wise gradient
0.4
0.3
0.2
0.1
0
50
Continuous vs step-wise
100
150
Elution volume
200
Adapted from Pharmacia: Ion Exchange Chromatography – Principles and Methods
連續梯度 Continuous gradient
Protein concentration
■
連
續
與
階
段
梯
度
比
較
NaCl
0.2
0.1
0
20
40
60
80
100
0.4
0.3
0.2
0.1
0
20
Effect of salt concentration
40
60
Elution volume
80
100
Adapted from Pharmacia: Ion Exchange Chromatography – Principles and Methods
0.3
Protein concentration
■
鹽
濃
度
比
較
100
0
100
0.5 M
Resolution improved
but protein diluted
50
0
100
0.5 M
50
0
0
Effect of elution volume
100
200
Elution volume
300
Adapted from Pharmacia: Ion Exchange Chromatography – Principles and Methods
0.5 M NaCl
50
Relative absorption
■
溶
離
體
積
會
影
響
解
析
度
B
5 cm column
Protein concentration
■
管
柱
長
短
的
影
響
1.0
0.5
A
0.03
0.04
0.06
0.1
0.2
B
10 cm column
A
0.03
0.04
1.0
0.5
0.06
0.1
Acetate concentration (M)
Effect of column length
0
0.2
0
Sample
applied
Salt gradient elution begins
Salt elutes and brings the proteins moving forward,
but proteins are adsorbed again in the front and retarded
Salt gradient of ion exchange can concentrate the proteins in sample elution
Adapted from Scope RK (1987) Protein Purification – Principles and Practice p.112
■ 離子交換的梯度溶離有濃縮效果
■ 離子交換法操作要點 Summary on operation
Equilibration
Used gel should be regenerated and equilibrated well
Elution
Eluted with NaCl in continuous or step-wise gradient
Gradient
Use proper concentration and volume in elution
Buffer pH
Keep target proteins in weak charged state
Non-specific
Wash out contaminants with low NaCl concentration
Sample
Equilibrated in buffer, don’t overload column capacity
Elute through
Flow target protein through and adsorb others
Dead volume
Eliminate any dead volume in the column
Juang RH (2005) EPA
■ 離子交換法實例 Two-step cellulase purification
FPLC (fast performance liquid chromatography)
0.5
0.2 M NaCl
0.5
Mono Q HR 5/5
1 mL/min
20 mM Tris, pH 7.6
Crude cellulase 2.5 mg
Q
0
10
+
-
20
S
0.5 M
NaCl
30
+
+
Mono S HR 5/5
1 mL/min
20 mM acetate,
pH 3.6
0
Retention time (min)
Adapted from Pharmacia (1991) Ion Exchange Chromatography – Principles and Methods p.127
10
20
0.4
Protein content / Activity
■
離
子
交
換
法
實
例
DEAE Sepharose CL-6B
SDS-PAGE
0.3
SS
NaCl
0.2
0.3
0.2
0.1
0.1
0
0
A typical example
100
200
Elution volume
300
Juang RH (2005) EPA
■ 色層焦集法如何拉出 pH 梯度
6
9
8
7
6
9
9
9
● Polybuffer contains ampholyte which is a
mixture of chemicals having continuous pKa
9
8
7
6
?
實際結果
pH
● Polybuffer 含有連續 pKa 的緩衝分
子 (ampholyte) 可拉出連續 pH 梯度
無法拉出 pH 梯度
Buffering
effect
假如含有許
多緩衝分子
8
9
8
7
6
6
If there are many
buffering molecules
in the buffer…..
Volume
How chromatofocusing creates its pH gradient
7
6
Volume
Juang RH (2005) EPA
3.3.5 色層焦集法 Chromatofocusing
其介質也是一種離子交換介質
但所使用的 Polybuffer 可拉出穩定的 pH 梯度
Chromatofocusing using ion exchange bead, but eluted with Polybuffer
Juang RH (2005) EPA
5
Polybuffer
■
色
析
焦
集
法
的
焦
集
機
制
6
7
++
+
+
++
+
+
+ 0 焦集在等電點
+
在低 pH 處帶正電被排斥
Protein at lower pH is positively
charged and repelled by the beads
Protein focused at its pI
超越等電點 (帶負電) 被吸附
8
Focusing mechanism in chromatofocusing
Protein moves beyond its pI will be
negatively charged and retarded
Juang RH (2005) EPA
■
環
境
影
響
分
子
的
帶
電
性
質
Buffer pH
10
9
8
7
Isoelectric point,
pI
+
6
5
4
3
0
Net Charge of a Protein
Juang RH (2005) EPA
● 抹香鯨肌紅蛋白 (pI = 8.2) 追過 馬肌紅蛋白 (pI = 7.4)
A molecule might be caught up by another protein in chromatofocusing
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